Following the initial discovery of two legume-nodulating Burkholderia strains (L. Moulin, A. Munive, B. Dreyfus, and C. Boivin-Masson, Nature 411:948–950, 2001), we identified as nitrogen-fixing legume symbionts at least 50 different strains of Burkholderia caribensis and Ralstonia taiwanensis, all belonging to the-subclass of proteobacteria, thus extending the phylogenetic diversity of the rhizobia. R. taiwanensis was found to represent 93% of the Mimosa isolates in Taiwan, indicating that-proteobacteria can be the specific symbionts of a legume. The nod genes of rhizobial-proteobacteria (-rhizobia) are very similar to those of rhizobia from the-subclass (-rhizobia), strongly supporting the hypothesis of the unique origin of common nod genes. The-rhizobial nod genes are located on a 0.5-Mb plasmid, together with the nifH gene, in R. taiwanensis and Burkholderia phymatum. Phylogenetic analysis of available nodA gene sequences clustered-rhizobial sequences in two nodA lineages intertwined with-rhizobial sequences. On the other hand, the-rhizobia were grouped with free-living nitrogen-fixing-proteobacteria on the basis of the nifH phylogenetic tree. These findings suggest that-rhizobia evolved from diazotrophs through multiple lateral nod gene transfers